Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

DEPhT: a novel approach for efficient prophage discovery and precise extraction

Advances in genome sequencing have produced hundreds of thousands of bacterial genome sequences, many of which have integrated prophages derived from temperate bacteriophages. These prophages play key roles by influencing bacterial metabolism, pathogenicity, antibiotic resistance, and defense against viral attack. However, they vary considerably even among related bacterial strains, and they are challenging to identify computationally and to extract precisely for comparative genomic analyses. Here, we describe DEPhT, a multimodal tool for prophage discovery and extraction. It has three run modes that facilitate rapid screening of large numbers of bacterial genomes, precise extraction of prophage sequences, and prophage annotation. DEPhT uses genomic architectural features that discriminate between phage and bacterial sequences for efficient prophage discovery, and targeted homology searches for precise prophage extraction. DEPhT is designed for prophage discovery in Mycobacterium genomes but can be adapted broadly to other bacteria. We deploy DEPhT to demonstrate that prophages are prevalent in Mycobacterium strains but are absent not only from the few well-characterized Mycobacterium tuberculosis strains, but also are absent from all ∼30 000 sequenced M. tuberculosis strains.     

Chromosome replication in an Escherichia coli dnaA mutant integratively suppressed by prophage P2.

Escherichia coli CRT4624-P2sig5 is a dnaA mutant in which integration of the prophage P2sig5 has occurred at the attP2II site (min 85). This strain was integratively suppressed, and when cells were shifted to 42 degrees C replication was initiated at a site in or near the P2 prophage. Initially, this replication occurred primarily in the direction that corresponds to the clockwise direction on the genetic map. Replication also occurred in the counterclockwise direction, but the initiation of replication in this direction occurred approximately 40 min later than the initiation of replication in the other direction. Because of this delay, the replication forks that traveled in the clockwise direction were the first to arrive in the region of the replication terminus. These replication forks ceased replication near the aroD locus (min 37), and it is proposed that the replication terminus is between the aroD and rac loci (min 31). A model is proposed for the cycle of chromosome replication in this strain at 42 degrees C.     

Distribution of Gifsy-3 and of Variants of ST64B and Gifsy-1 Prophages amongst Salmonella enterica Serovar Typhimurium Isolates: Evidence that Combinations of Prophages Promote Clonality

Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.     

Integration defective mutants of bacteriophage P2

Summary Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.Exceptional lysogens carrying two or more int prophages may be obtained: they may produce spontaneously even more phage than normal lysogens, and they segregate out defective, singly lysogenic clones at low frequency. These exceptional lysogens carry both prophages in location I, presumably in tandem.Strains carrying two or more int prophages but defective in phage production were also isolated. One of these carries its prophages at two different, not closely linked, chromosomal locations.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112

D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.     

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.     

Evolution along the parasitism-mutualism continuum determines the genetic repertoire of prophages

Integrated into their bacterial hosts’ genomes, prophage sequences exhibit a wide diversity of length and gene content, from highly degraded cryptic sequences to intact, functional prophages that retain a full complement of lytic-function genes. We apply three approaches—bioinformatics, analytical modelling and computational simulation—to understand the diverse gene content of prophages. In the bioinformatics work, we examine the distributions of over 50,000 annotated prophage genes identified in 1384 prophage sequences, comparing the gene repertoires of intact and incomplete prophages. These data indicate that genes involved in the replication, packaging, and release of phage particles have been preferentially lost in incomplete prophages, while tail fiber, transposase and integrase genes are significantly enriched. Consistent with these results, our mathematical and computational approaches predict that genes involved in phage lytic function are preferentially lost, resulting in shorter prophages that often retain genes that benefit the host. Informed by these models, we offer novel hypotheses for the enrichment of integrase and transposase genes in cryptic prophages. Overall, we demonstrate that functional and cryptic prophages represent a diversity of genetic sequences that evolve along a parasitism-mutualism continuum.     

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.     

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for antirepressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage).Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.     

The role of prophage for genome diversification within a clonal lineage of Lactobacillus johnsonii: characterization of the defective prophage LJ771

Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (“antitoxin”)/pemK (“toxin”) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5′ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.     

Excisionase in Pf filamentous prophage controls lysis‐lysogeny decision‐making in Pseudomonas aeruginosa

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis‐lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5′‐untranslated regions overlap. XisF4 and Pf4r not only auto‐activate their own expression but also repress each other. Furthermore, two H‐NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.     

Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine

Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.     

Indirect SOS induction is promoted by ultraviolet light-damaged miniF and requires the miniF lynA locus

Indirect prophage induction is produced by transfer to recipients of u.v.-damaged F plasmid (95 kb). We tested whether the SOS signal can be produced by miniF, a 9.3 kb restriction fragment, coding for the replication and segregation functions of plasmid F. We used λminiF, a hybrid phage-plasmid. u.v.-irradiated λminiF induced prophages φ80 or λ and sfiA, a chromosomal SOS gene, in more than 50% of the infected cells. The maximal inducing dose produced about 0.5 pyrimidine dimers per kb and left 1% of λminiF survivors. Thus, the SOS signal produced by u.v.-damaged λminiF was almost as potent as that resulting from direct u.v.-irradiation of the lysogens. The u.v.-damaged vector λ, devoid of miniF, failed to promote SOS induction. In contrast, efficient induction was observed when u.v.-damaged λminiF infected a λ immune host, in which replication and expression of the phage genome were repressed. When replication and expression of the miniF genome was repressed by Hfr incompatibility, SOS induction was largely prevented. All these facts indicate that, in the hybrid λ-miniF, it is the u.v.-damaged miniF that generates an SOS signal.To locate on the miniF genome the loci that are involved in the production of the SOS signal, we isolated deletions spanning all the miniF restriction fragments. We characterized six mutant phenotypes (Par⁺, Rep^?, Fid^?, Par-2, Par-1 and SOS^?) related to four functions; partition, copy number, replication and SOS induction. A locus, we call lynA, 800bp long, located by deletion mapping between the two origins of replication oriP and oriS is required for the production of an inducing signal.We postulate that indirect SOS induction by u.v.-damaged miniF results from the disturbance of the lynA function that may be involved in the co-segregation of F plasmid with the host chromosome.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

Packaging Specific Segments of the Salmonella Chromosome with Locked-in Mud-P22 Prophages

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.     

ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli

The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acid-starved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp⁰). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp⁰ bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.